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Intersectional genetics system for stem cell selection
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Resumo(s)
Stem cell therapy holds promise for treating conditions like macular degeneration, stroke, and diabetes.
In most of these therapies, replenishing a target cell type is desired. In contrast, the undifferentiated cells
or differentiated cells of the wrong lineage are unnecessary or can lead to side effects such as teratoma
formation. One way to overcome this is to engineer the transplanted cells to carry a functional genetic
cell ablation system in all transplanted cells except for the target cell type. Versatile Entry Codes (VEnCodes) based on the intersection of regulatory elements can enable selective gene delivery to specific
cell types. This study explores two alternative approaches to eliminate undesired cells while safeguarding cells of interest selectively: an inducible Caspase 9 (iCasp9)-mediated cell death system and a VEnCode-compatible split-Cre recombinase (Split-Cre-VEnCode) system. The iCasp9 system used the TetOn system with rtTA as the input and iCasp9 as the output. By precisely controlling iCasp9 expression
levels and activity using doxycycline and the chemical inducer of dimerization AP20187, respectively,
the system successfully induced cell death in HEK-293T cells. Whereas further testing of the drug
AP20187 dosage is required to increase cell death, these promising results warrant future investigations
in combination with the VEnCode system and different cell types to assess this system's potential applications fully. The Split-Cre-VEnCode system utilized LoxP sites strategically placed to manipulate
downstream gene expression. Successful DNA recombination events were observed by monitoring the
expression of mScarlet-I as a reporter gene. The system demonstrated inversion and deletion capabilities,
offering controlled on/off effects. However, optimization is still required to split the Cre recombinase
into >2 non-complementing fragments to unlock the full capabilities of this Split-Cre-VEnCode system.
While this study primarily evaluated the systems in HEK-293T cells, future studies should validate their
functionality in diverse cell lines. Additionally, long-term effects, stability, kinetics of activation, and
potential off-target effects need to be investigated. These findings highlight the potential of these ablation
systems complemented with VEnCode-based biosensor prototypes to enhance the specificity and safety
of genetic delivery systems, providing powerful tools for conditional gene therapy and precise cellular
ablation in biomedical research. iCasp9-mediated cell death and split-Cre recombinase systems are potential tools for cell ablation and targeted gene expression.
Descrição
Tese de mestrado, Biologia Molecular e Genética, 2023, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Caspase 9 indutível Split-Cre morte celular terapias celulares sistema VEnCode Teses de mestrado - 2023